S226: LOSS OF NR4A1 IS ASSOCIATED WITH HIGHER EXPRESSION OF IMMUNE CHECKPOINT COMPONENTS AND REDUCED T CELL-MEDIATED LYMPHOMA CELL KILLING IN AGGRESSIVE LYMPHOMA
Authors: K. Pansy, K. Fechter, A. Arra, M. Szmyra, S. Haingartner, A. Ramsay, H. Greinix, C. Beham-Schmid, P. Neumeister, A. Deutsch
Journal: HemaSphere (2022)

Background: Aggressive lymphomas represent the most common type of lymphoid malignancies with a five-year survival rate of 60%. Despite effective initial treatment, one-third of all patients will experience a relapse, warranting more research to discover novel therapeutic strategies. We recently detected a significant lower expression of NR4A1 that correlates with a poor lymphoma-specific survival. Furthermore, ectopic expression of NR4A1 induces apoptosis in vitro and suppresses lymphoma growth in xenografts indicating its tumor suppressive properties. Aims: The aim of this study was to comprehensively study the function of Nr4a1 loss in lymphomagenesis. Methods: Therefore, we intercrossed the EµMyc lymphoma mouse model with the Nr4a1-/- mouse and monitored them until the onset of disease. Furthermore, we transplanted lymphoma cells of EµMyc Nr4a1-/- and EµMyc Nr4a1+/+ mice into immune-competent C57BL/6 mice and immune-deficient Fox Chase SCID beige mice. Furthermore, we determined the expression levels of immune checkpoint components in biopsies of our human diffuse large B cell lymphoma (DLBCL) cohort and correlated them to NR4A1 content. Finally, to investigate the immune-regulatory function of Nr4a1, we performed co-culture cytotoxicity assays using OVA257-264 peptide-pulsed EµMyc Nr4a1+/+ and EµMyc Nr4a1-/- lymphoma cells and Ctla4+/+ and Ctla4-/- OVA targeting CD8+ T cells derived from OT-I mice and measured T cell-mediated lymphoma cell lysis via flow cytometry, respectively. Results: We observed that the loss of Nr4a1 leads to an accelerated lymphomagenesis in vivo, concomitant with increased expression of immune checkpoint components of the Pd1-Pdl1-Pdl2- and Ctla4-Cd80-Cd86-axes. Immuno-competent, but not immune-deficient mice, transplanted with Nr4a1-deficient lymphoma cells exhibited rapid lymphoma development, reduced survival, and upregulation of the immune checkpoint components like in the primary model. Importantly, low NR4A1 expression correlated with high expression of immune checkpoint components in biopsies of our human DLBCL cohort largely resembling our mouse data. To unravel the impact of Nr4a1 on T cell-mediated lymphoma cell killing and on the regulation of the Ctla-4-Cd80-Cd86-axis of aggressive lymphomas, we performed co-culture cytotoxicity assays using OVA peptide-pulsed EµMyc Nr4a1+/+ and EµMyc Nr4a1-/- lymphoma cells and Ctla4+/+ and Ctla4-/- OVA targeting CD8+ T cells. In these experiments, we observed a massively diminished lymphoma cell killing in the EµMyc Nr4a1-/- setting, when we used Ctla4+/+ OT-1 CD8+ T cells. Interestingly, when we used Ctla4-/- OT-1 CD8+ T cells for the cytotoxicity assays, lymphoma cell killing was enhanced in the Nr4a1-/- setting compared to that using Ctla4+/+ OT-1 CD8+ T cells. Image:Summary/Conclusion: Our data suggest that Nr4a1 plays a critical role in regulating the licensing of immune evasion in aggressive lymphomas by regulating immune checkpoint expression. Thus, it might act as a promising target to restore anti-lymphoma immune responses.